The physical properties of plasma membrane lipids play vital roles in B cell receptor (BCR) activation, yet remarkably little is known regarding how lipids regulate the organization and activity of proteins within this signaling pathway. Overcoming this basic knowledge gap is necessary to elucidate the mechanisms underlying this essential biological function and will lead to the development of novel interventions for immune- related diseases. The experiments outlined in this proposal will identify the role plasma membrane lipids in regulating interactions between the BCR and down-stream mediators of the immune response, and will develop experimental methods to modulate lipid-mediated interactions in cells. The working hypothesis is that healthy B cells exploit heterogeneity maintained by a critical composition of its membrane lipids, to balance interactions between the BCR, activating kinases, and down-regulating phosphatases in order to facilitate early activation steps and suppress receptor activity in resting cells. The proposed research tests this working hypothesis by quantifying lipid-mediated interactions between the BCR and plasma membrane proteins in normal B cells and in cells treated with targeted perturbations of lipid heterogeneity. Guided by extensive preliminary data, two specific aims will be pursued: 1) Quantify and modulate effective interactions between the BCR and minimal membrane anchors and 2) Identify the roles of lipids in early B cell receptor activation. Both aims will utilize a new and tested method developed in the applicant's laboratory to directly measure interaction potentials between plasma membrane proteins using super-resolution imaging techniques. Both aims will also apply a novel class of membrane perturbations shown to modify the phase behavior of isolated plasma membrane vesicles and the composition of cross-linked BCR receptor clusters in cells. In the first aim, interaction energies will be measured between the BCR and membrane protein anchor motifs expressed in B cells and interactions will be modulated with biochemical perturbations. Under the second aim, the activity of full length proteins involved in BCR signaling will be measured and their effective interactions with other signaling components will be quantified over a range of experimental conditions. A predictive model will be developed that includes protein and lipid interactions, and plasma membrane heterogeneity will be investigated in lymphoma cells with known defects in BCR signaling. Although the proposed research will take place in B cells, it has implications for studying of role of lipids in other biological proceses and cell types. This approach is innovative because it draws on cutting edge experimental methodologies as well as the unique perspective that lipids impact functional processes by modulating effective interactions between embedded proteins. The proposed work is significant because it will establish a mechanism for lipid-mediated control of immune signaling processes enabling new strategies for the treatment of immune related disease through manipulation of plasma membrane physical properties.